Oligonucleotide analogues

Abstract

The present invention relates to novel bicyclic and tricyclic nucleoside and nucleotide analogues as well as to oligonucleotides comprising such elements. The nucleotide analogues, LNAs (Locked Nucleoside Analogues), are able to provide valuable improvements to oligonucleotides with respect to affinity and specificity towards complementary RNA and DNA oligomers. The novel type of LNA modified oligonucleotides, as well as the LNAs as such, are useful in a wide range of diagnostic applications as well as therapeutic applications. Among these can be mentioned antisense applications, PCR applications, strand displacement oligomers, as substrates for nucleic acid polymerases, as nucleotide based drugs, etc. The present invention also relates to such applications.

1. A method for using an oligomer to isolate, purify, amplify, detect, identify, quantify or capture a natural or synthetic nucleic acid, the oligomer being modified with at least one Locked Nucleic Acid (LNA), the method comprising the step of contacting LNA-modified oligomer with the nucleic acid under conditions sufficient to form a binding complex to isolate, purify, amplify, detect, identify, quantify or capture the nucleic acid, wherein the LNA is represented by the following general formula I: 
       
         
           
           
               
               
           
         
       
       wherein X is selected from —O—;
 B is selected from hydrogen, hydroxy, optionally substituted C 1-4 -alkoxy, optionally substituted C 1-4 -alkyl, optionally substituted C 1-4 -acyloxy, nucleobases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands; 
 P designates a 5′-terminal group optionally including the substituent R 5 ; 
 one of the substituents R 2 , R 2* , R 3 , and R 3*  is a group P* which designates an internucleoside linkage or a 3′-terminal group; 
 the substituents R 4* , and R 2* , together designating the biradical —(CR*R*) r —O—(CR*R*) s —, each R* is independently selected from hydrogen, halogen, hydroxy, mercapto, amino, optionally substituted C 1-6 -alkoxy, optionally substituted C 1-6 -alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, and r is 0 (zero) and s is 1, or r is 1 and s is 0 (zero); and 
 each of the substituents R 1* , R 2 , R 3 , R 5 , and R 5* , which are present and not involved in P or P* is independently selected from hydrogen, optionally substituted C 1-12 -alkyl, optionally substituted C 2-12 -alkenyl, optionally substituted C 2-12 -alkynyl, hydroxy, C 1-12 -alkoxy, C 2-12 -alkenyloxy, carboxy, C 1-12 -alkoxycarbonyl, C 1-12 -alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C 1-6 -alkyl)amino, carbamoyl, mono- and di(C 1-6 -alkyl)-amino-carbonyl, amino-C 1-6 -alkyl-aminocarbonyl, mono- and di(C 1-6 -alkyl)amino-C 1-6 -alkyl-aminocarbonyl, C 1-6 -alkylcarbonylamino, carbamido, C 1-6 -alkanoyloxy, sulphono, C 1-6 -alkylsulphonyloxy, nitro, azido, sulphanyl, C 1-6 -alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted; 
 and basic salts and acid addition salts thereof, wherein a natural or synthetic nucleic acid is isolated, purified, amplified, detected, identified, quantified, or captured using the oligomer. 
 
     
     
       2. The method of  claim 1 , wherein the oligomer comprises 1 to 1000 nucleosides of the formula I and 0-1000 nucleosides selected from naturally occurring nucleosides and nucleotide analogues, with the proviso that the sum of the number of nucleosides and the number of LNA(s) is at least 2. 
     
     
       3. The method of  claim 1 , wherein the oligomer comprises a photochemically active group, a thermochemically active group, a chelating group, a reporter group, or a ligand. 
     
     
       4. The method of  claim 3 , wherein the method further comprises the step of immobilizing the oligomer to a solid support. 
     
     
       5. The method of  claim 3 , wherein the photochemically active group, thermochemically active group, chelating group, reporter group, or the ligand includes a spacer (K), said spacer comprising a chemically cleavable group. 
     
     
       6. The method of  claim 3 , wherein the photochemically active group, the thermochemically active group, the chelating group, the reporter group, or the ligand is attached via the biradical of at least one of the LNA(s) of the oligomer. 
     
     
       7. The method of  claim 1 , wherein the method further comprises the steps of capturing and detecting the binding complex as being indicative of presence of a double stranded or single stranded nucleic acid. 
     
     
       8. The method of  claim 7 , wherein the nucleic acid is RNA or DNA. 
     
     
       9. The method of  claim 1 , wherein the method further comprises the step of purifying the nucleic acid from the binding complex. 
     
     
       10. The method of  claim 9 , wherein the nucleic acid is RNA or DNA. 
     
     
       11. The method of  claim 1 , wherein formation of the binding complex is conducted by one of in-situ hybridisation, Southern hydridisation, Dot blot hybridisation, reverse Dot blot hybridisation, or by Northern hybridisation. 
     
     
       12. The method of  claim 1 , wherein the binding complex serves as a template for a nucleic acid sequencing reaction, primer extension reaction, or nucleic acid amplification reaction. 
     
     
       13. The method of  claim 12 , wherein the nucleic acid amplification reaction is an essentially linear reaction. 
     
     
       14. The method of  claim 12 , wherein the nucleic acid amplification reaction is an essentially exponential reaction. 
     
     
       15. The method of  claim 12 , wherein the binding complex serves as a template for a nucleic acid amplification reaction, the method further comprising the step of producing a double stranded DNA product comprising at least one single stranded end. 
     
     
       16. The method of  claim 1 , wherein at least one of the LNA groups comprises a nucleobase as the substituent B. 
     
     
       17. The method of  claim 1 , wherein one of the substituents R 3  and R 3*  designates P*. 
     
     
       18. The method of  claim 1 , wherein one or more nucleosides have the following formula Ia 
       
         
           
           
               
               
           
         
       
       wherein P, P*, B, X, R 1* , R 2 , R 2* , R 3 , R 3* , R 4* , R 5 , and R 5*  are as defined in  claim 1 . 
     
     
       19. The method of  claim 18 , wherein R 3*  designates P*. 
     
     
       20. The method of  claim 19 , wherein the oligomer comprises one biradical constituted by two non-geminal substituents. 
     
     
       21. The method of  claim 1 , wherein R 3*  designates P*. 
     
     
       22. The method of  claim 21 , wherein R 2  is selected from hydrogen, hydroxy, and optionally substituted C 1-4 -alkoxy, and R 3* , R 3 , R 5 , and R 5*  designate hydrogen. 
     
     
       23. The method of  claim 22 , wherein B is selected from nucleobases. 
     
     
       24. The method of  claim 23 , wherein B is selected from adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7-deazaxanthine, 7-deazaguanine, N 4 ,N 6 -ethanocytosin, N 6 ,N 6 -ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C 3 -C 6 )-alkynylcytosine, 2,6-diaminopyrimidino, 2,6-diaminopyrazine, 1-methyl-pyrazolo[4,3-d]pyrimidino-5,7 (4H,6H)-dione, 1-methyl-pyrazolo[4,3-d]pyrimidine-5,7 (4H,6H)-dione, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanin, and inosine. 
     
     
       25. The method of  claim 1 , wherein any internucleoside linkage within the oligomer is selected from linkages consisting of 2 to 4 groups/atoms selected from —CH 2 —, —O—, —S—, —NR H —, >C═O, >NR H , >C═S, —Si(R″) 2 —SO—, —S(O) 2 —, —P(O) 2 —, —P(O,S)—, —P(S) 2 —, —PO(R″)—, —PO(OCH 2 )—, and —PO(NHR H )—, where R H  is selected from hydrogen and C 1-4 -alkyl, and R″ is selected from C 1-6 -alkyl and phenyl. 
     
     
       26. The method of  claim 25 , wherein the internucleoside linkage is selected from —CH 2 —CH 2 —CH 2 —, —CH 2 —CO—CH 2 —, —CH 2 —CHOH—CH 2 —, —O—CH 2 —O—, —O—CH 2 —CH 2 —, —O—CH 2 —CH═, —CH 2 —CH 2 —O—, —NR H —CH 2 —, —CH 2 —CH 2 —NR H —, —CH 2 —NR H —CH 2 —, —O—CH 2 —CH 2 —NR H —, —NR H —CO—O—, —NR H —CO—NR H —, —NR H —CS—NR H —, NR H —C(═NR H )—NR H —, —NR H —CO—CH 2 —NR H —, —O—CO—O—, —O—CO—CH 2 —O—, —O—CH 2 —CO—O—, —CH 2 —CO—NR H —, —O—CO—NR H —, —NR H —CO—CH 2 —, —O—CH 2 —CO—NR H —, —O—CH 2 —CH 2 —NR H —, —CH═N—O—, —CH 2 —NR H —O—, —CH 2 —O—N═, —CH 2 —O—NR H —, —CO—NR H —CH 2 —, —CH 2 —NR H —O—, —CH 2 —NR H —CO—, —O—NR H —CH 2 —, —O—NR H —, —O—CH 2 —S—, —S—CH 2 —O—, —CH 2 —CH 2 —S—, —O—CH 2 —CH 2 —S—, —S—CH 2 —CH═, —S—CH 2 —CH 2 —, —CH 2 —CH 2 —O—, —S—CH 2 —CH 2 —S—, —CH 2 —S—CH 2 —, —CH 2 —SO—CH 2 —, —CH 2 —SO 2 —CH 2 —, —O—SO—O—, —S(O) 2 —O—, —O—S(O) 2 —CH 2 —, —O—S(O) 2 —NR H —, —NR H —S(O) 2 —CH 2 —, —O—S(O) 2 —CH 2 —, —O—P(O) 2 —O—, —O—P(O,S)—O—, —O—P(S) 2 —O—, —S—P(O) 2 —O—, —S—P(O,S)—O—, —S—P(S) 2 —O—, —O—P(O) 2 —S—, —O—P(O,S)—S—, —O—P(S) 2 —S—, —S—P(O) 2 —S—, —S—P(O,S)—S—, —S—P(S) 2 —S—, —O—PO(R″)—O—, —O—PO(OCH 3 )—O—, —O—PO(BH 3 )—O—, O—PO(NHR N )—O—, —O—P(O) 2 —NR H —, —NR H —P(O) 2 —O—, —O—P(O,NR H )—O—, and —O—Si(R″) 2 —O—. 
     
     
       27. The method of  claim 26 , wherein the internucleoside linkage is selected from —CH 2 —CO_NR H —, —CH 2 —NR H —O—, —S—CH 2 —O—, —O—P(O) 2 —, —O—P(O,S)—O—, —O—P(S) 2 —O—, —NR H —P(O) 2 —O—, —O—P(O,NR H )—O—, —O—PO(R″)—O—, —O—PO(CH 2 )—O—, and —O—PO(NHR N )—O—, where R H  is selected from hydrogen and C 1-4 -alkyl, and R″ is selected from C 1-6 -alkyl and phenyl. 
     
     
       28. The method of  claim 1 , wherein each of the substituents R 1* , R 2 , R 3 , R 3* , R 5 , and R 5*  of the one or more LNA nucleosides, which are present and not involved in P or P* is independently selected from hydrogen, optionally substituted C 1-6 -alkyl, optionally substituted C 2-6 -alkenyl, hydroxy, C 1-6 -alkoxy, C 2-6 -alkenyloxy, carboxy, C 1-6 -alkoxycarbonyl, C 1-6 -alkylcarbonyl, formyl, amino, mono- and di(C 1-6 -alkyl)amino, carbamoyl, mono- and di(C 1-6 -alkyl)-amino-carbonyl, C 1-6 -alkyl-carbonylamino, carbamido, azido, C 1-6 -alkanoyloxy, sulphono, sulphanyl, C 1-6 -alkylthio, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, and halogen, where two geminal substituents together may designate oxo, and where R N* , when present and not involved in a biradical, is selected from hydrogen and C 1-4 -alkyl. 
     
     
       29. The method of  claim 1 , wherein each of the substituents R 1* , R 2 , R 1 , R 3* , R 5 , and R 5*  of the LNA(s) which is present and not involved in P or P* designates hydrogen. 
     
     
       30. The method of  claim 1 , wherein P is a 5′-terminal group selected from hydrogen, hydroxy, optionally substituted C 1-6 -alkyl, optionally substituted C 1-6 -alkoxy, optionally substituted C 1-6 -alkylcarbonyloxy, optionally substituted aryloxy, monophosphate, diphosphate, triphosphate, and -W-A′, wherein W is selected from —O—, —S—, and —N(R H )— where R H  is selected from hydrogen and C 1-6 -alkyl, and where A′ is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands. 
     
     
       31. The method of  claim 1 , wherein P* is a 3′-terminal group selected from hydrogen, hydroxy, optionally substituted C 1-6 -alkoxy, optionally substituted C 1-6 -alkylcarbonyloxy, optionally substituted aryloxy, and -W-A′, wherein W is selected from —O—, —S—, and —N(R H )— where R H  is selected from hydrogen and C 1-6 -alkyl, and where A′ is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands. 
     
     
       32. The method of  claim 1 , wherein the oligomer corresponds to the following formula V:
   G-[Nu-L] n(0) -{[LNA-L] m(q) -[Nu-L] n(q) } q -G*  V 
 
       wherein
 q is 1-50; 
 each of n(0), . . . , n(q) is independently 0-10000; 
 each of m(1), . . . , m(q) is independently 1-10000; 
 with the proviso that the sum of n(0), . . . , n(q) and m(1), . . . , m(q) is 2-15000; 
 G designates a 5′-terminal group; 
 each Nu independently designates a nucleoside selected from naturally occurring nucleosides and nucleoside analogues; 
 each LNA independently designates a nucleoside analogue; 
 each L independently designates an internucleoside linkage between two groups selected from Nu and LNA, or L together with G* designates a 3′-terminal group; and 
 each LNA-L independently designates a nucleoside analogue of the general formula I 
 
       
         
           
           
               
               
           
         
       
       wherein the substituents B, P, P*, R 1* , R 2 , R 2* , R 3 , R 4* , R 5 , and R 5* , and X are as defined in  claim 1 . 
     
     
       33. The method of  claim 1 , wherein the oligomer further comprises a PNA mono- or oligomer segment of the formula 
       
         
           
           
               
               
           
         
       
       wherein B is a defined above for the formula I, AASC designates hydrogen or an amino acid side chain, t is 1-5, and w is 1-50. 
     
     
       34. The method of  claim 1 , wherein the oligomer has an increased specificity towards complementary ssRNA or ssDNA compared to a corresponding reference oligomer which does not contain any LNA units. 
     
     
       35. The method of  claim 1 , wherein the oligomer has an increased affinity towards complementary ssRNA or ssDNA compared to a corresponding reference oligomer which does not contain any LNA units. 
     
     
       36. The method of  claim 1 , wherein the oligomer is capable of binding to a target sequence in a dsDNA or dsRNA molecule by of strand displacement or by triple helix formation. 
     
     
       37. The method of  claim 1 , wherein the oligomer is more resistant to nucleases than a corresponding reference oligomer which does not contain any LNA units. 
     
     
       38. The method of  claim 1 , wherein the oligomer has nucleic acid catalytic activity. 
     
     
       39. The method of  claim 1 , wherein the oligomer comprises one or more 2′-O,4′-C-linked nucleoside units. 
     
     
       40. A kit for the isolation, purification, amplification, detection, identification, quantification, or capture of a natural or synthetic nucleic acid, the kit comprising a reaction body, at least one of the oligomers produced by  claim 1 ; and directions for using the kit. 
     
     
       41. The method of  claim 1 , wherein the oligomer comprises one or more nucleosides of the general formula I wherein:
 the substituents R 4* , and R 2* , together designating the biradical —(CR*R*) r —O—(CR*R*) s —, each R* is independently selected from hydrogen, halogen, hydroxy, mercapto, amino, optionally substituted C 1-6 -alkoxy, optionally substituted C 1-6 -alkyl, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, and r is 0 (zero) and s is greater than 1, or r is greater than 1 and s is 0 (zero). 
 
     
     
       42. The method of  claim 1 , wherein the oligomer comprises one or more nucleosides of the general formula I wherein:
 B designates a nucleobase; 
 P, optionally together with R 5 , designates a bond to the terminal end of an internucleoside linkage to a succeeding monomer, or a 5′-terminal group selected from hydrogen, hydroxy, optionally substituted C 1-6 alkyl, optionally substituted C 1-6 -alkoxy, optionally substituted C 1-6 -alkylcarbonyloxy, optionally substituted aryloxy, monophosphate, diphosphate, triphosphate, and -W-A′, wherein W is selected from —O—, —S—, and —N(RH)— where RH is selected from hydrogen and C 1-6 -alkyl, and where A′ is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where photochemically active groups is selected from quinones, diazirines, aromatic azides, benzophenones, psoralens, diazo compounds, and diazirino compounds, and thermochemically active groups is selected from carboxylic acids, carboxylic acid esters, carboxylic acid halides, carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemic arbazides, aldehydes, ketones, primary alkohols, secondary alkohols, tertiary alkohols, phenols, alkyl halides, thiols, disulphides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, and boronic acid derivatives; 
 one of the substituents R 3 , and R 3*  is a group P* which designates a bond to the terminal end of an internucleoside linkage to a preceding monomer, or a 3′-terminal group selected from hydrogen, hydroxy, optionally substituted C 1-6 -alkoxy, optionally substituted C 1-6 -alkylcarbonyloxy, optionally substituted aryloxy, and -W-A′, wherein W is selected from —O—, —S—, and —N(RH)—where RH is selected from hydrogen and C 1-6 -alkyl, and where A′ is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where photochemically active groups is selected from quinones, diazirines, aromatic azides, benzophenones, psoralens, diazo compounds, and diazirino compounds, and thermochemically active groups is selected from carboxylic acids, carboxylic acid esters, carboxylic acid halides, carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alkohols, secondary alkohols, tertiary alkohols, phenols, alkyl halides, thiols, disulphides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, and boronic acid derivatives; 
 the substituents R 2*  and R 4*  together designates a biradical selected from —CH 2 —O—, —NH—CH 2 —, —N(CH 3 )—CH 2 — AND —S—CH 2 —; 
 each of the substituents R 1* , R 2 , R 3 , R 3* , R 5 , and R 5*  which are not involved in P, or P*, designate hydrogen.